Virus protein antigens of the JC virus

ABSTRACT

The invention concerns new virus-like particles composed of the VP1 protein of the JC virus, a process for their production and their use in analytical, diagnostic and therapeutic methods.

DESCRIPTION

The present invention concerns new virus-like particles and their use inanalytical, diagnostic and therapeutic methods.

The JC virus (JCV) belongs to the group of human polyoma viruses. JCVcan cause a sub-acute demyelinizing disease of the brain by a lyticinfection of myelin-forming oligodendrocytes and an abortive infectionof astrocytes. This infection, which is referred to clinically asprogressive multifocal leukoencephalopathy (PML), leads to the formationof demyelinizing foci in the cerebrum cerebellum and brain stem andusually ends lethally within a few months.

With PML there is usually no significant humoral or cellular immuneresponse to JCV which makes it difficult to diagnose the disease.Although JCV appears to be present in about 80% of the adult population,PML generally only develops in connection with a weakening of the immunesystem. The increasing use of immuno-suppressive drugs and theincreasing number of HIV-infected patients has led to a considerableincrease in PML diseases in recent years. According to currentestimations a PML develops in about 2-5% of AIDS patients.

The known methods of diagnosis for detecting a PML disease essentiallycomprise image forming methods such as CT (computer tomography) and MRI(magnetic resonance imaging) as well as immunocytochemical methods basedon biopsies or autopsies. Recently PCR detection methods have increasedin importance, virus DNA amplification from cerebrospinal fluid (CSF)yielding reliable and specific results (Weber et al., J. Infect. Dis.(1994), 1138-1141 and McGuire et al., Annals of Neurology 37 (1995),395-399).

The disadvantages of the diagnostic methods known from the state of theart are that they can only be carried out with a large amount of workand are therefore unsuitable for a reliable routine diagnosis. This alsoapplies to PCR methods in which there is a high contamination risk thatcan lead to a falsification of the results. It would therefore bedesirable to have available a method and reagents which allow a reliablediagnosis of PML in a more simple manner. Furthermore there is a needfor an agent that can be used for the therapeutic treatment of PMLdiseases.

Hence the object of the invention is to provide methods and reagentswith which the above-mentioned goals can be achieved and which at leastpartially avoids the disadvantages of the state of the art.

It was surprisingly found that the virus protein 1 (VP1) of the JC virusis an excellent agent for the immunochemical detection of JCV.Furthermore it was found that VP1 can be produced in a large yield inthe form of virus-like particles (VLP) by recombinant DNA techniques.

Hence one subject matter of the invention is a virus-like particle (VLP)which is composed of several molecules of the virus protein 1 (VP1), themain structural protein of the JC virus. This VLP is in particularcharacterized in that it has a structure that reacts immunologicallywith anti-JCV antisera and is free of nucleic acids associated with JCV.

It was surprisingly found that, after purification, recombinant VP1 ofJCV can associate to form virus-like particles (VLP). Such VLP have anicosahedral structure with a diameter of about 50 nm. The advantage ofVLP over an individual VP1 protein is above all that the propertiesor/and effects of the natural virus are more exactly simulated and thata broader field of application is opened up. Further advantages over thenatural virus are, in particular, increased work safety and it wassurprisingly also found that the VLP has a higher immunogenicity thanthe purified total virus.

The VP1 of JCV is the main structural protein of the capsid envelope ofJCV e.g. from wild-type strains or mutagenized strains of JCV. In aspecial embodiment the VLP is composed of recombinantly produced VP1. Inthis case the term VP1 also encompasses proteins which differ fromwild-type VP1 by mutations such as substitutions, insertions or/anddeletions. In order to produce recombinant VP1 one preferably uses anucleic acid which comprises the sequence shown in SEQ ID NO.1, asequence corresponding to this within the scope of the degeneracy of thegenetic code or a sequence hybridizing with it under stringentconditions wherein the nucleic acid sequence or a recombinant vectorcontaining this sequence is introduced into a suitable host cell, thehost cell is cultured under conditions in which an expression of thenucleic acid sequence takes place and the protein is isolated from thecell or cell supernatant. Stringent hybridization conditions arepreferably defined according to Sambrook et al. (1989) Molecular CloningA Laboratory Manual, Second Edition, Cold Spring Harbor LaboratoryPress, and comprise a wash step of 30 min in 0.1×SSC, 0.5% SDS at 68° C.

The VLP according to the invention can furthermore have one or severaladditional heterologous proteins in the capsid structure. This meansthat a heterologous protein is anchored in the capsid structure, atleast part of this protein being preferably accessible from outside. Inprinciple all proteins are suitable as this heterologous protein whichcan be incorporated into the capsid structure and do not impair theself-assembly of the VLP. For example it may be desirable to provide theVLP with an antigenic determinant by means of the heterologous proteinthat can be detected by a specific antibody. On the other hand theheterologous protein can be a binding partner for a cell surfacereceptor which enables an interaction of the VLP with a specific classor subclass of cells which carry the corresponding receptor. Afterbinding to the receptor such an interaction is for example thetriggering of a particular signal or the internalization of the VLP.

The origin of the proteins is not limited to a particular organism and,depending on the special type of application, it is possible toincorporate eukaryotic, in particular human proteins, and alsoprokaryotic or viral proteins into the VLP. Examples of suitableproteins are surface proteins or membrane proteins, explicit examples ofwhich are CD4 for a human cell surface receptor and the envelope proteinof an immuno-deficiency virus e.g. HIV-gp 120 for a viral surfaceprotein. It goes without saying that the heterologous protein can bemodified if necessary, e.g. shortened, using recombinant methodsespecially if the natural protein cannot be incorporated into the VLP orimpairs the self-assembly.

In a further special embodiment the VLP can contain one or severalactive substances inside the capsid structure. In this description anactive substance is understood as any molecule which is not usuallypresent in the medium used for self-assembly. Such active substancesinclude for example macromolecules such as nucleic acids i.e. RNA, DNAor artificial modified nucleic acids as well as proteins and otherphysiologically active substances that can be of a natural, synthetic orrecombinant type. Examples of such physiologically active substances aree.g. lipids, phospholipids, peptides, drugs, toxins etc.

Yet a further subject matter of the present invention is a nucleic acidwhich codes for a VP1 protein and comprises

(a) the nucleotide sequence shown in SEQ ID NO. 1

(b) a nucleotide sequence corresponding to the sequence from (a) withinthe scope of the degeneracy of the genetic code or/and

(c) a nucleotide sequence hybridizing with one of the sequences from (a)or/and (b) under stringent conditions.

The nucleic acid according to the invention is preferably located on arecombinant vector, in particular under the control of an expressionsignal. Examples of suitable vectors are described in Sambrook et al.,Supra, chapters 1, 2, 3, 4, 16 and 17. The invention also concerns acell which is transformed with a vector according to the invention.

A further aspect of the invention also concerns a process for theproduction of a VLP in which VP1 is purified and converted into a formin which an assembly of several VP1 molecules to form a VLP takes place.Suitable conditions for assembly are for example present when VP1 ispurified from a cell culture supernatant after a differential cushioncentrifugation over sucrose and metrizamide.

The VLP is preferably produced recombinantly in which case a nucleicacid coding for a VP1 protein is introduced into a cell, the transformedcell is cultured in a medium under conditions in which an expression ofthe nucleic acid takes place and the expression product is isolated fromthe cell or from the medium. The recombinant VP1 is isolated directlyfrom the host cells or/and the cell culture supernatant depending on thehost/vector system used.

The advantage of the recombinant process is above all that VLP can beobtained in a simple manner, in high purity and in large amounts. Theuse of baculoviruses together with insect cells, e.g. with the insectcell line Sf 158, has proven to be an effective expression system inpractice.

In order to produce VLP which have incorporated a heterologous proteinwithin the capsid structure or VLP which contain an active substanceinside the capsid structure, the above-mentioned production process ismodified by adding the heterologous proteins or/and active substances inthe desired amount or concentration at a suitable time point i.e. beforeassembly of the VLP and subsequently allowing the assembly. In thismanner VLP can form which have incorporated a heterologous protein intothe capsid envelope or/and contain an enclosed active substance, e.g. anucleic acid, inside. Heterologous polypeptides can for example beincorporated into the capsid envelope by recombinant co-expression ofthe respective polypeptides i.e. of the VP1 polypeptide and of theheterologous polypeptide in a suitable host cell e.g. a eukaryotic cell.Active substances can be incorporated into the interior of the capsidenvelope by for example dissociation of the capsid envelope andsubsequent re-association in the presence of the active substance or byosmotic shock of the VLP in the presence of the active substance.

Yet a further aspect of the present invention concerns a method for theimmunological determination of specific antibodies to JCV in a sample inwhich the antibodies are detected qualitatively or/and quantitatively bybinding to a VLP composed of VP1 or to a component thereof. For reasonsof ready availability it is preferable to use VLP composed ofrecombinant VP1 molecules.

A large number of test formats for such immunological methods ofdetermination are known to a person skilled in the art and therefore donot have to be individually elucidated. In general such methods arecarried out by contacting the VLP or components thereof with the sampleunder suitable conditions in order to allow binding of specificantibodies against the JC virus to the VLP or components thereof,separating the immune complexes formed from other sample components anddetecting the presence of antibodies. For this one can for example useVLP which are adsorptively or covalently coupled to a solid phase orcoupled via a suitable binding partner.

The antibody to be detected that is bound to the virus protein afterincubation with the sample liquid is subsequently detected by a specificreceptor directed against the antibody to be detected e.g. a detectionantibody, protein A or an antigen capable of binding to the antibodye.g. a VLP. The exact type of this receptor is not critical and it isfor example possible to use polyclonal or monoclonal detectionantibodies such as anti-human antibodies from various animal speciessuch as rabbits, mice or goats or antibodies that are specificallydirected against the Fc part of immunoglobulins or immunoglobulinclasses.

The receptor capable of binding to the antibody to be detectedadditionally includes an agent that allows the detection i.e. a labelthat is directly or indirectly associated with the receptor e.g. bymeans of specific binding pairs such as biotin/avidin. Such labels arealso known to a person skilled in the art and for example includeradioactivity, enzymes, luminescent or fluorescent labels. Aparticularly preferred test format is the ELISA in which a receptor withan enzyme label is used.

The above-mentioned immunological method of determination can be used todiagnose progressive multifocal leukoencephalopathy (PML) ifcerebrospinal fluid (CSF) is used as the sample. In a preferredembodiment a parallel determination is carried out to diagnose PML inwhich CSF and serum from the same person are used as samples, both thesesamples being preferably collected in parallel. In order to obtainwell-founded results it is particularly preferable to standardize themeasured values relative to the total immunoglobulin concentration. Asuitable reference parameter is the so-called antibody specificity index(ASI) which is defined as the ratio of the titre of specific antibodiesto JCV in CSF and serum divided by the ratio of the Ig totalconcentration in CSF and serum. It has turned out that an ASI value of≧1.5 for a positive diagnosis of PML gives reliable results.

Hence a further subject matter of the present invention is a test kitfor the determination of specific antibodies to JCV which comprises, ina separate spatial arrangement, VLP of JCV or components thereof, anagent for the detection of antibodies and optionally conventionalbuffers and auxiliary substances. The test kit is preferably suitablefor the detection of human antibodies. The agent for the detection ofantibodies includes in particular a receptor capable of bindingspecifically to the antibody to be detected which is provided directlyor indirectly with a detectable label. The test kit can also contain thenecessary substrates and detection substances.

The VLP according to the invention can also be used therapeutically e.g.for the treatment of PML. Hence the VLP according to the invention aresuitable for the production of a therapeutic or/and prophylactic vaccinewhich is used against an infection with JCV. In this regard a VLP shouldbe used in particular which neither contains heterologous proteinsincorporated into the capsid structure nor encloses active substancesinside. By this means it is possible, on the one hand, to saturatereceptors on the surface of the oligodendrocytes in order to at leastslow down further infection and, on the other hand, to stimulate thehumoral immune response to JCV. The cellular immune response which canbe detected by an antigen-specific T cell proliferation can also bestimulated by administering recombinant VP1. However, modified VLP canalso be used alternatively which then have preferably incorporated abinding partner for a surface receptor of the corresponding targetcells, i.e. in particular of oligodendrocytes, into the capsid structureand have enclosed at least one active substance in the interior whichprevents the multiplication or spreading of the virus. In this case theactive substance is preferably a virostatic agent such as cytosinearabinoside.

In a further embodiment the VLP can be used as a transport vehicle. SuchVLP contain a suitable active substance enclosed in their interior andpreferably a heterologous protein incorporated into the capsid structurewhich is capable of binding to a specific surface receptor of thedesired target cell. This ensures the specificity of the interactionwith the intended target cells and can be adapted to numerous cell typesdepending on the use. An example of such a use is the site-directedtransport of TNF-α antisense nucleic acids to oligodendrocytes inmultiple sclerosis since it is known that in this disease a surge ofTNF-α expression leads to demyelinization. Another example is the use ofVLP as a transporter system for nucleic acids in gene therapy.

Therefore a further subject matter of the invention is a pharmaceuticalcomposition comprising a VLP as well as optionally conventional buffers,auxiliary agents, additives or/and diluents. The VLP in this case can bean unmodified VLP or a VLP with a heterologous protein incorporated intothe capsid structure or/and with an active substance enclosed in theinterior.

The invention is now further elucidated by the following examples aswell as by the attached figures and sequence protocols.

SEQ ID NO. 1 shows the nucleotide sequence of a DNA sequence isolatedfrom JCV which codes for VP1; SEQ ID NO. 2 shows the corresponding aminoacid sequence; SEQ ID NO. 3 shows an amplification primer used toisolate the VP1 coding sequence; SEQ ID NO. 4 shows a furtheramplification primer and Brief Description of Drawings: FIG. 1 shows aschematic representation of the working steps to prepare recombinantVP1; FIG. 2 shows the packaging of heterologous DNA in VP1-VLP, FIG. 3shows the immunoreactivity of an anti-VP1-VLP (Parts A-E) immune serumand FIG. 4 shows the neutralization of JCV by an anti-VP1-VLP immuneserum.

EXAMPLES Example 1

Recombinant production of JCV-VP1

JCV-DNA (strain Mad-4) was isolated from SVG cells and the sequencecoding for the VP1 gene was amplified by means of PCR. The followingprimers were used for the PCR which were provided with a BamHI andHindIII restriction cleavage site for the subsequent cloning.

Primer 1 (SEQ ID NO. 3): 5′-GTA CGG GAC TGC AGC ACC TGC TCT TGA AG-3′Primer 2 (SEQ ID NO. 4): 5′-TAC AAT AAA AGC TTT TGA TTA CAG CAT TT-3′

In the PCR reaction 100 pmol of each primer was used in a reactionvolume of 50 μl. The DNA was preamplified in a 50 μl mixture by 20amplification cycles comprising 45 s denaturation at 94° C., 45 sannealing at 55° C. and 45 s extension at 70° C. The denaturation wasextended to 5 min in the first cycle. Each subsequent extension wasincreased by 1 s and the last cycle was extended to 5 min. 10 μl of thisreaction was mixed with the primers and 2.0 U Taq DNA polymerase(Pharmacia, Freiburg, Germany) and subjected to a PCR under the aboveconditions for a total of 25 cycles.

The PCR products were purified by agarose gel electrophoresis, cleavedwith BamHI and HindIII and ligated into the cloning site of aBamHI/HindIII cleaved p-BlueBac-III vector (Invitrogen, Netherlands) toobtain the vector pBlueBac-VP containing the VP1 gene. Aftertransformation of E.coli cells with pBlueBac-VP and identification ofpositive clones, the recombinant plasmids were isolated and transfectedinto Sf 158 insect cells with wild-type baculovirus DNA.

After passaging the supernatant twice on Sf 158 cells, the recombinantbaculoviruses were purified (cf. FIG. 1).

The recombinant VP1 was expressed in the Sf 158 host cells and secretedinto the cell supernatant at a concentration of 0.9 mg protein per ml.The expression was detected by Western blot of cell culture supernatantsusing a rabbit anti-SV40 hyperimmune serum.

It was possible to purify the VP1 to homogeneity from the cell culturesupernatant by means of differential cushion centrifugation over sucroseand metrizamide.

It can be shown by electron microscopic investigations that the VP1associated to form VLP that have an icosahedral structure with adiameter of about 50 nm and a density of ca. 1.31 g/ml in CsClgradients. Protein biochemical investigations of the VP1 contained inthe VLP by means of mass spectroscopy after tryptic digestion yieldedthe expected fragment masses. The recombinant VLP proved to be highlyimmunogenic after immunization in rabbits.

Example 2

Immunological Determination of Specific Antibodies to the JC Virus inSerum and CSF

Recombinant VP1 was titrated out against the hyperimmune serum to SV40from rabbits and antisera of PML patients. The optimal antigenconcentration was determined as 200 ng protein of the product obtainedafter differential cushion centrifugation corresponding to ˜70 ng VP1per well. CSF total protein was determined by means of trichloroaceticacid precipitation. Immunoglobulin G and albumin in the serum and CSFwere determined immunochemically by nephelometry. Intrathecal antibodysynthesis was analysed according to the methods of Reiber and Lange(Clin. Chem. 37 (1991), 1153-1160) and Weber et al., (J. Immunol.Methods 136 (1991), 133-137). Intrathecal synthesis of VP1-specificantibodies was defined as a ratio of the antibody titre in CSF and serum(ASI) of at least 1.5.

In order to carry out the determination 200 ng of the cell culturesupernatant of Sf 158 cells infected with pBlueBac-III-VP1 was appliedto each well of a microtitre plate with 96 wells (Grainer Frickenhausen,Germany) at 4° C. for 12 to 14 hours. The unspecific binding wassaturated by incubation for 1 hour at 37° C. with 200 μl 5% Blotto perwell. Serum was diluted in Blotto starting at 1:500 in log2 steps andthe highest dilution used was 1:80,000. CSF was diluted in Blottostarting at 1:5 in log2 steps and the highest dilution used was 1 to40,000. After binding the primary antibody for 3 hours at 37° C., theplates were washed seven times with 150 mM PBS containing 0.05% Tween20. Bound antibody was detected by incubation for 2 hours at 37° C. witha peroxidase-conjugated anti-human IgG antiserum from rabbits (JacksonLaboratories Dianova, Hamburg, Germany). After washing seven times withPBS, the peroxidase-conjugated antibody was visualized usingo-phenylene-diamine. After 15 minutes the reaction was stopped with 1.3N sulfuric acid and the optical density (OD) was measured at 495/620 nmin a Titertek MC340 MKII-ELISA reader. The antibody titre in CSF andserum was calculated using EXEL™.

In selected cases a Western blot analysis of the intrathecal immuneresponse was carried out. After electrophoretic separation of 1 μg VP1per lane in a 12% SDS-PAGE gel, the antigen was transferredelectrophoretically onto a HiBond-PVDF membrane (Amersham Buchler KG,Germany). The unspecific antibody binding was saturated for 1 hour at37° C. with 5% Blotto. Serum or CSF antibodies were added to each laneat an IgG concentration of 10 mg/l and incubated for 3 hours at 37° C.After three washes in PBS the membranes were incubated with aperoxidase-conjugated anti-human IgG antiserum from rabbits (JacksonLaboratories Dianova, Hamburg). Bound detection antibodies werevisualized according to the manufacturer's instructions on Hyperfilm-ECLusing an ECL kit (Amersham Buchler KG, Germany).

Example 3

Diagnostic Determination of PML

Parallel CSF/serum samples from 189 patients were examined. Intrathecalsynthesis of anti-VP1 antibodies was found in 28/34 (82%) of the PMLpatients at an ASI ≧1.5 whereas an ASI ≧1.5 was only found in 5/155 ofthe control patients. Serum antibodies to VP1 were found in all PMLpatients as well as in 28/37 (76%) of the multiple sclerosis patients,43/50 (86%) of the control group, 29/33 (88%) of patients with impairedblood-brain barrier function and 31/35 (89%) of the HIV-positivepatients.

The intrathecal synthesis of anti-VP1 antibodies was checked by Westernblot analysis in individual cases. An almost identical intensity in theCSF and serum was obtained in PML patients with a VP1 ASI <1.5 whereas 4sera from patients with ASI in the range 12-107 exhibited very weakserum bands and considerably stronger CSF bands.

A statistical analysis of the antibody specificity indices (ASI) wascarried out in the 5 different patient groups. In the normal group theaverage VP1 ASI was 0.92 (±0.16, range 0.65-1.26), in the multiplesclerosis groups it was 0.91 (±0.38, range 0.34-2.49), in the group withimpaired blood-brain barrier function it was 0.92 (±0.22, range0.52-1.36), in the HIV-positive group it was 4.35 (±12.7, range0.35-67.2) and in the PML group it was 12.59 (±24.42, range 0.38-107).Using the Kruskal-Wallis test a significant difference is found betweenthe 5 groups (P<0.0001). Using the Mann-Whitney test there is asignificant difference between the PML group and the control group, thegroup with impaired blood-brain barrier function and the multiplesclerosis group (P<0.0001) and between the PML group and theHIV-positive group at P<0.001. The differences between the other 6 grouppairs were not significant.

The above results show that an examination of the intrathecal synthesisof antibodies directed against JCV-VP1 is a suitable test for thediagnosis of PML. In the investigation carried out a sensitivity of 82%and a specificity of 96% was found using a statistically significantVP1-ASI of ≧1.5. Although the ELISA assay thus has a somewhat lowersensitivity than a PCR assay with nested primers (93%) it represents auseful addition to current test procedures since it is morecost-effective, simpler to carry out and less susceptible tofalse-positive results by contamination. Important applications includethe screening and examinations of numerous samples, the rapiddetermination of unequivocally positive samples as well as the exclusionof unequivocally negative samples since according to the present resultsa negative antibody test in serum excludes the diagnosis of PML.

Example 4

Detection of a Cellular Immune Response to Recombinant VP1

It was possible to detect a cellular immune response to recombinantJCV-VP1 in a healthy test person and a PML patient. The antigen-specificproliferation was dose-dependent. An antigen-specific T cellproliferation was detected at a final concentration of 1-10 μg antigenwhich reached its maximum on the 6th day. This experiment indicates thathealthy test persons and PML patients also have a cellular immuneresponse to JCV and in particular to the VP1 protein in addition to ahumoral response.

Example 5

Packaging of foreign proteins and exogenous DNA in VLP of the mainstructural protein VP1 of JCV

5.1 Packaging of viral envelope glycoproteins

In order to package a foreign protein VP1 and the envelope glycoproteingp160 of the ape immunodeficiency virus SIVmac32H were co-expressed in amodel experiment. In both cases the vector pBlueBac already mentioned inexample 1 was used as the base vector for the expression.

For this Sf158 cells were infected at a multiplicity (number ofinfectious units per cell) of 5 with the baculoviruses recombinant forVP1 and at a multiplicity ratio of gp160 to VP1 of 0.2, 0.5 and 2. Fivedays after the co-infection the cells and the cell culture supernatantswere examined by Western blot analysis for the presence of gp160 andVP1. In this examination it was possible to detect the efficientcoexpression of gp160 and VP1 with an antibody mixture directed againstgp160 and VP1. It was also possible to detect gp160 and VP1 separately.In this case a multiplicity ratio of 2 yielded the best coexpressionresult. The association of gp160 with the VP1-VLP was demonstrated afterpelleting the cell culture supernatant through a 40% sucrose cushion. Itwas possible to detect gp160 separately in the resulting pellet as wellas gp160 and VP1 by means of the antibody mixture.

5.2. Packaging of exogenous DNA

Within the framework of developing efficient transfer systems for genetherapy it was examined whether exogenous DNA can be packaged into theVP1-VLP. Three approaches were investigated for this. The purifiedVP1-VLP were suspended in TBS/Ca (10 mM Tris-HCl pH: 7.5, 150 mM NaCl,0.01 mM CaCl₂), H₂O redistilled or dissociation buffer (TBS pH: 8.5containing 10 mM EGTA and 5 mM DTT). Exogenous DNA in the form of apUC18 derivative which carries the 1.2 kB long V1/V2 region of the envgene for the envelope glycoprotein of HIV-1han2 whose sequence has beenpublished by Sauermann et al. (AIDS Research and Human Retroviruses 6(1990), 813-823) (total vector ca. 4.1 kB) was added at a weight ratioof DNA to VP1-VLP of 1:10 and 1:20. The incubation was carried out for60 minutes at 37° C. The reaction mixture carried out in TBS/Ca was thensubjected to an osmotic shock and incubated for a further 30 minutes at37° C. All samples were then dialysed overnight against reassociationbuffer (TBS pH: 7.5 containing 1 mM CaCl₂). Subsequently all sampleswere digested intensively with DNase I, diluted with TBS/Ca andultracentrifuged overnight through a 40% sucrose cushion. Afterresuspension of the particles, the DNA was extracted and the V1/V2region (ca. 1.8 kB) was amplified by PCR by means of specific primersand the amplificate was separated in an agarose gel. In this experimentit was found that exogenous DNA could be packaged into the VP1-VLP (FIG.2) using all three methods (lanes 1 to 6). The exogenous DNA wasefficiently protected against digestion with DNase I after packaging.The best packaging efficiency was reached at a DNA to VP1-VLP ratio of1:20 and after a sequence of dissociation and reassociation (lane 6).

According to an estimate based on the positive control it was possibleto package about 3 to 4 g exogenous DNA into 80 g purified VP1-VLP.

Example 6

Development of a Therapeutic Vaccine Against the Human Polvomavirus JCVBased on VLP of the Main Structural Protein VP1

In order to produce a therapeutic vaccine against the human polyomavirusJCV the main structural protein VP1 was expressed in insect cells withthe aid of recombinant baculoviruses (example 1). After expression VP1forms typical virus-like particles (VLP). The VLP represent a mixture ofempty and full particles with a diameter of about 50 to 60 nm. TheVP1-VLP were purified to homogeneity and exhibited a floating density inCsCl gradients of 1.32 g/ml (empty particle) and 1.34 g/ml (fullparticle).

Immunogenicity of VP1-VLP

In order to examine the immunogenicity 100 μg of the purified VP1-VLPwere mixed with 300 μg haemocyanine from the keyhole limpet, emulsifiedin complete Freund's adjuvant and a rabbit was intramuscularly immunizedtherewith. A booster immunization was carried out four weeks later withincomplete Freund's adjuvant. The analysis of the titre of anti-VP1antiserum in an ELISA at various collection dates after the lastimmunization of the rabbit is shown in FIG. 3A. 50 ng of the purifiedVP1-VLP was absorbed to an ELISA plate (Greiner, Nurtingen) for theinvestigations and the anti-VP1 antiserum of the correspondingcollection dates (+: 7 weeks; *: 13 weeks, □: 15 weeks; X: 20 weeks; ⋄:25 weeks) were titrated out at the stated dilutions. The end point titrewas defined as the dilution at which the reactivity corresponded to thatof the preimmunization serum (−). As can be seen in FIG. 3A the immuneserum had reached an end point titre of about 10⁵ six weeks after thelast immunization.

The specificity of the anti-VP1 antiserum was examined in a Western blot(WB). For this about 20 ng purified antigen per lane was separated bymeans of SDS polyacrylamide gel electrophoresis and transferred onto anitrocellulose membrane. In FIG. 3B the VPS1-VLP were used as theantigen and the reactivity of the anti-VP1 antiserum from variouscollection dates (lane 2: 7 weeks; lane 3: 13 weeks; lane 4: 15 weeks;lane 5: 20 weeks; lane 6: 25 weeks) was compared with the reactivity ofa preimmunization serum (lane 1) and with an anti-SV40 hyperimmune serum(lane 7). In FIG. 3C purified VP1-VLP were also used whereas in FIG. 3Bpurified natural JCV particles and in FIG. 3E purified natural SV40particles were used as the antigen. In figures C-E each lane Prepresents the preimmunization serum, each lane 1 represents theanti-SV40 immune serum and each lane 2 represents the anti-VPSantiserum.

The specificity of the antiserum was as expected. As shown by a Westernblot analysis only VP1-VLP-specific proteins were recognized and aconsiderable increase of the reactivity depending on the bleeding timepoint after the last immunization was observed (FIG. 3B, lanes 2 to 6).The reactivity was comparable with an anti-SV40 hyperimmune serum (lane7). The immunoreactivity of the anti-VP1-VLP immune serum was alsotested against various antigens. The hyperimmune serum additionallyrecognized the VP1 of SV40 (FIG. 3D, lane 1) and the VP1 of natural JCV(FIG. 3E, lane 1) in addition to the recombinant VP1 (FIG. 3C, lane 1).Again the reactivity was comparable with that of the anti-SV40 immuneserum (FIGS. 3C to E, lane 2 in each case).

Binding and Inhibition of the Binding of the VP1-VLP to SVG Cells

For these experiments the VP1-VLP were labelled with ¹²⁵I. Firstly theratio of the ¹²⁵I-VP1-VLP and SVG cells was determined which wereobtained by transfection of human foetal glial cells with a SV40 mutantwith a defect in the replication of origin (Major et al., Proc. Natl.Acad. Sci. USA 82 (1985), 1257-1261). A saturation binding was achievedwith 2×10⁶ cpm ¹²⁵I-VP1-VLP and 10⁵ SVG cells. The binding inhibition ofthe anti-VP1-VLP immune serum was examined under these test conditions.The ¹²⁵I-VP1-VLP binding was suppressed by 95% at serum dilutions of 1:5and 1:10 and a binding inhibition of about 75% was still observed evenat a serum dilution of 1:20. Whereas a binding inhibition of only 20%was measurable at a serum dilution of 1:40, no binding inhibition wasachieved at a serum dilution of 1:80.

Neutralization of JCV

A new test was developed to examine the neutralization capacity of theVP1-VLP hyperimmune serum. This test is based on the intracellulardetection of VP1 after infection of SVG cells with JCV. As can be seenin FIG. 4 a complete neutralization of JCV was achieved at a dilution ofthe JCV infection stock of 1:160 and a serum dilution of 1:24 since noVP1 was detectable. At a serum dilution of 1:40 VP1 was recognized butin a reduced form which indicates a partial JCV neutralization. Thisalso applies to a serum dilution of 1:80, whereas at a serum dilution of1:160 no neutralization effects were achieved in comparison to acontrol. Similar results were also obtained at a dilution of the JCVinfection stock of 1:80.

These results show that the VP1-VLP induce the immune response whichwould be expected of a vaccine with a potential for success.Neutralizing and binding inhibiting antibodies were induced. Moreoverthe VP1-VLP are also able to induce a proliferative T cell reactivity inthe peripheral blood lymphocytes of JCV-positive but healthy individuals(examples 2-4).

4 1 1121 DNA JC virus 1 gtacgggact gcagcacctg ctcttgaagc atatgaagatggccccaaca aaaagaaaag 60 gagaaaggaa ggaccccgtg caagttccaa aacttcttataagaggagga gtagaagttc 120 tagaagttaa aactggggtt gactcaatta cagaggtagaatgcttttta actccagaaa 180 tgggtgaccc agatgagcat cttaggggtt ttagtaagtcaatatctata tcagatacat 240 ttgaaagtga ctccccaaat agggacatgc ttccttgttacagtgtggcc agaattccac 300 tacccaatct aaatgaggat ctaacctgtg gaaatatactcatgtgggag gctgtgacct 360 taaaaactga ggttataggg gtgacaagtt tgatgaatgtgcactctaat gggcaagcaa 420 ctcatgacaa tggtgcaggg aagccagtgc agggcaccagctttcatttt ttttctgttg 480 ggggggaggc tttagaatta cagggggtgc tttttaattacagaacaaag tacccagatg 540 gaacaatttt tccaaagaat gccacagtgc aatctcaagtcatgaacaca gagcacaagg 600 cgtacctaga taagaacaaa gcatatcctg ttgaatgttgggttcctgat cccaccagaa 660 atgaaaacac aagatatttt gggacactaa caggaggagaaaatgttcct ccagttcttc 720 atataacaaa cactgccaca acagtgttgc ttgatgaatttggtgttggg ccactttgca 780 aaggtgacaa cttatacttg tcagctgttg atgtctgtggcatgtttaca aacaggtctg 840 gttcccagca gtggagagga ctctccagat attttaaggtgcagctaagg aaaaggaggg 900 ttaaaaaccc ctacccaatt tctttccttc ttactgatttaattaacaga aggactccta 960 gagttgatgg gcagcctatg tatggcatgg atgctcaagtagaggaggtt agagtttttg 1020 agggaacaga ggagcttcca ggggacccag acatgatgagatacgttgac aaatatggac 1080 agttgcagac aaaaatgctg taatcaaaag cttttattgt a1121 2 354 PRT JC virus 2 Met Ala Pro Thr Lys Arg Lys Gly Glu Arg LysAsp Pro Val Gln Val 1 5 10 15 Pro Lys Leu Leu Ile Arg Gly Gly Val GluVal Leu Glu Val Lys Thr 20 25 30 Gly Val Asp Ser Ile Thr Glu Val Glu CysPhe Leu Thr Pro Glu Met 35 40 45 Gly Asp Pro Asp Glu His Leu Arg Gly PheSer Lys Ser Ile Ser Ile 50 55 60 Ser Asp Thr Phe Glu Ser Asp Ser Pro AsnArg Asp Met Leu Pro Cys 65 70 75 80 Tyr Ser Val Ala Arg Ile Pro Leu ProAsn Leu Asn Glu Asp Leu Thr 85 90 95 Cys Gly Asn Ile Leu Met Trp Glu AlaVal Thr Leu Lys Thr Glu Val 100 105 110 Ile Gly Val Thr Ser Leu Met AsnVal His Ser Asn Gly Gln Ala Thr 115 120 125 His Asp Asn Gly Ala Gly LysPro Val Gln Gly Thr Ser Phe His Phe 130 135 140 Phe Ser Val Gly Gly GluAla Leu Glu Leu Gln Gly Val Leu Phe Asn 145 150 155 160 Tyr Arg Thr LysTyr Pro Asp Gly Thr Ile Phe Pro Lys Asn Ala Thr 165 170 175 Val Gln SerGln Val Met Asn Thr Glu His Lys Ala Tyr Leu Asp Lys 180 185 190 Asn LysAla Tyr Pro Val Glu Cys Trp Val Pro Asp Pro Thr Arg Asn 195 200 205 GluAsn Thr Arg Tyr Phe Gly Thr Leu Thr Gly Gly Glu Asn Val Pro 210 215 220Pro Val Leu His Ile Thr Asn Thr Ala Thr Thr Val Leu Leu Asp Glu 225 230235 240 Phe Gly Val Gly Pro Leu Cys Lys Gly Asp Asn Leu Tyr Leu Ser Ala245 250 255 Val Asp Val Cys Gly Met Phe Thr Asn Arg Ser Gly Ser Gln GlnTrp 260 265 270 Arg Gly Leu Ser Arg Tyr Phe Lys Val Gln Leu Arg Lys ArgArg Val 275 280 285 Lys Asn Pro Tyr Pro Ile Ser Phe Leu Leu Thr Asp LeuIle Asn Arg 290 295 300 Arg Thr Pro Arg Val Asp Gly Gln Pro Met Tyr GlyMet Asp Ala Gln 305 310 315 320 Val Glu Glu Val Arg Val Phe Glu Gly ThrGlu Glu Leu Pro Gly Asp 325 330 335 Pro Asp Met Met Arg Tyr Val Asp LysTyr Gly Gln Leu Gln Thr Lys 340 345 350 Met Leu 3 29 DNA JC virus 3gtacgggact gcagcacctg ctcttgaag 29 4 29 DNA JC virus 4 tacaataaaagcttttgatt acagcattt 29

What is claimed is:
 1. A non-infectious virus-like particle, whereinsaid particle comprises several molecules of the virus protein VP1 ofthe JC virus as the only JCV component in the virus like particle. 2.The particle according to claim 1, wherein said VP1 is recombinant VP1.3. The particle according to claim 1, wherein said VP1 is coded by anucleic acid comprising (a) the nucleotide sequence shown in SEQ ID NO:1(b) a nucleotide sequence which encodes the amino acid sequenceaccording to SEQ ID NO:2, and (c) a nucleotide sequence hybridizing withone of the sequences from (a)-(b) under conditions that comprise a washstep of 30 minutes in 0.1×SSC, 0.5% SDS at 68 C.
 4. The particleaccording to claim 1, wherein said particle has at least one additionalheterologous protein incorporated into the capsid structure.
 5. Theparticle according to claim 4, wherein the additional protein is a cellsurface receptor or a binding partner for a cell surface receptor. 6.The particle according to claim 5, wherein the additional protein is aviral surface protein.
 7. The particle according to claim 5, wherein theadditional protein is gp120 from HIV.
 8. The particle according to claim1, wherein said particle contains at least one active substance insidethe capsid structure.
 9. The particle according to claim 8, wherein theactive substance is selected from the group consisting of nucleic acids,proteins and physiologically active substances.
 10. The particleaccording to claim 9, wherein the active substance comprises a nucleicacid.
 11. A process for the production of a non-infectious virus-likeparticle, wherein said particle comprises several molecules of the virusprotein VP1 of the JC virus as the only JCV component in the virus likeparticle, comprising purifying VP1, and assembling several VP1 moleculesto form a non-infectious virus like particle.
 12. The process accordingto claim 11, further comprising introducing a nucleic acid coding for aVP1 protein into a cell, culturing the transformed cell in a mediumunder conditions in which the nucleic acid is expressed, and isolatingthe expression product from the cell or from the medium.
 13. The processaccording to claim 12, wherein said nucleic acid coding for a VP1protein comprises (a) the nucleotide sequence shown in SEQ ID NO:1, (b)a nucleotide sequence encoding an amino acid sequence according to SEQID NO:2, and (c) a nucleotide sequence hybridizing with one of thesequences from (a)-(b) under conditions that comprise a wash step of 30minutes in 0.1×SSC, 0.5% SDS at 68° C.
 14. The process according toclaim 12, wherein the nucleic acid coding for a VP1 protein is presentin at least one copy on a recombinant vector.
 15. The process accordingto claim 14, wherein the expression of said VP1 protein is under thecontrol of an expression signal.
 16. The process according to claim 12,wherein said cell is an insect cell.
 17. The process according to claim11, wherein the assembly is carried out in the presence of at least oneadditional protein during which the protein is incorporated into anon-infectious virus-like particle comprising more than one VP1molecule.
 18. The process according to claim 11, wherein the assembly iscarried out in the presence of a further substance during which thesubstance is enclosed in a non-infectious virus-like particle comprisingmore than one VP1 molecule.
 19. The process according to claim 18,wherein said further substance is a nucleic acid.
 20. A method for theimmunological determination of specific antibodies against JCV in asample, comprising obtaining a sample from a patient, binding anyantibodies in said sample to a non-infectious virus like particle or acomponent thereof, wherein said particle comprises several molecules ofthe virus protein VP1 of the JC virus as the only JCV component in thevirus like particle, and detecting any antibody/VP1 complexesqualitatively, quantitatively or both qualitatively and quantitativelyas an indication of said specific antibodies against JCV.
 21. The methodaccording to claim 20, wherein said virus protein VP1 is recombinantVP1.
 22. The method according to claim 20, further comprising separatingany formed immune complexes from other sample components, and detectingthe presence of antibodies, wherein the virus like particles orcomponents thereof are contacted with said sample under suitableconditions in order to allow binding of specific antibodies against JCvirus to the virus like particles or to components thereof.
 23. Themethod according to claim 20, wherein said sample is cerebrospinal fluidand said immunological method of determination is used to detectprogressive multifocal leukoencephalopathy.
 24. The method according toclaim 23, further comprising carrying out a second determination using aserum sample from said patient, wherein said serum sample is collectedfrom said patient at about the same time as said cerebrospinal fluid.25. A test kit for the determination of specific antibodies against a JCvirus, comprising a) a non-infectious virus-like particle, wherein saidparticle comprises several molecules of the virus protein VP1 of the JCvirus as the only JCV component in the virus like particle, and b) anagent for the detection of antibodies.
 26. The test kit according toclaim 25, wherein said agent for the detection of antibodies comprises aspecific receptor which binds to the antibodies to be detected andsuitable detection agents.